Epithelial tryptic serine proteases regulate ion channels, activate receptors, degrade matrix during tumor invasion and activate pathogenic viruses by cell-surface proteolysis. However, the specific proteases involved in many of these processes remain undefined. Preliminary studies in this laboratory identified a new family of membrane-associated tryptic serine proteases (MTSPs) genes on chromosome 16p and explored expression of these proteases in the lung. MTSPs are closely related to CAPI, which activates epithelial sodium channels (ENaQ in kidney, and to mast cell tryptases, which activate protease-activated receptor-2 (PAR-2) among other functions, but the functions of MTSPs are not yet known. The long-term objective of these studies is to elucidate the physiologic functions of MTSPs and mechanisms regulating MTSP activity in airway and alveolar epithelial cells. The overall hypothesis of these studies is that MTSPs regulate ENaC and activate protease activated receptors by controlled proteolysis at the cell surface of airway and alveolar epithelium. The proposed studies focus on prostasin, a prototypic MTSP expressed on the surface of airway and alveolar epithelial cells. Aim 1 is to determine whether prostasin regulates ENaC and activates PAR-2 in lung epithelial cells. Aim 2 is to investigate mechanisms regulating prostasin expression in lung by identifying cell types expressing prostasin in normal lung and cystic fibrosis, asthma and lung cancer, by identifying key promoter elements directing expression in lung epithelial cells, and by determining mechanisms of membrane anchoring and release. Aim 3 is to identify novel serine proteases expressed in lung epithelial cells which regulate important epithelial functions. This project will employ native and heterologous coexpression in mammalian cells and Xenopus oocytes for electrophysiologic studies of ENaC regulation and studies of PAR-2 activation by prostasin. Immunohistochemistry in normal and diseased lung and promoter analysis will examine regulation of prostasin expression. Enzymatic release, metabolic labeling, and mutagenesis will be employed to study mechanisms and function of membrane anchoring. Novel serine proteases expressed in respiratory epithelial cells will be identified by homology cloning. The proposed research investigates novel cell-surface proteolytic mechanisms regulating ENaC and PAR-2 in airway and alveolar epithelium relevant to obstructive airways disease and diseases characterized by altered ion and fluid transport, such as ARDS and cystic fibrosis. The research and career development plan will prepare the candidate to become an independent investigator in lung biology.